Abstract Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients, a major cause of hospital-acquired pneumonia and is particulary problematic in burn wound infections. Hfq is a conserved global post-transcriptional regulator that is required for the virulence of P. aeruginosa. In other organisms Hfq is best known for its ability to promote the base-pairing between small regulatory RNAs (sRNAs) and their target transcripts. Hfq-promoted interaction between an sRNA and its mRNA target typically functions to repress translation of the target, although Hfq can also inhibit translation in an sRNA-independent fashion. We have found that in P. aeruginosa Hfq associates with hundreds of transcripts as they emerge from RNA polymerase. Because transcription and translation are coupled in bacteria, the interaction of Hfq with these nascent transcripts presumably allows this RNA-binding protein to exert its regulatory effects on translation at the earliest possible opportunity. In Aim 1 we propose to identify the targets of all the sRNAs that interact with Hfq in P. aeruginosa and determine whether these sRNAs frequently work in conjunction with Hfq on nascent transcripts. The interaction of Hfq with nascent transcripts has important implications for how transcript abundance can be controlled; specifically, Hfq- dependent inhibition of the translation of nascent transcripts would render them accessible to the transcription termination factor Rho or to ribonucleases. In Aim 1 we will determine whether Hfq reduces the abundance of those nascent transcripts it binds through effects on Rho-mediated transcription termination or through effects on RNA degradation. We have found that a second global post-transcriptional regulator in P. aeruginosa called RsmA also targets hundreds of nascent transcripts, including many of those that are targeted by Hfq. RsmA is a key regulator of the switch between acute and chronic infection phenotypes in P. aeruginosa and in Aim 2 we will investigate whether Hfq and RsmA modulate each other?s activities through competition or cooperation on common targets. Like Hfq, the binding of RsmA to target mRNA species typically inhibits their translation. In Aim 3 we propose to determine whether the interaction of RsmA with nascent transcripts influences their abundance through effects on Rho-mediated transcription termination or effects on RNA degradation. The experiments outlined in this proposal are expected to illuminate how Hfq and RsmA control gene expression through their widespread targeting of nascent transcripts. The co-transcriptional activities of these global post- transcriptional regulators have been underexplored and our proposed studies may have implications not only for how Hfq and RsmA impact the virulence of P. aeruginosa, but also for how their orthologs influence virulence gene expression in other pathogenic bacteria that contain them.